Reversible protein phosphorylation is a major mechanism for the coordinated control of many fundamental cellular functions in eukaryotic organisms, including metabolism, growth, and differentiation. The phosphorylation status, and consequently the activity, of specific target proteins is regulated by the opposing actions of protein kinases and protein phosphatases. Generally, these enzymes are specific either for serine/threonine or for tyrosine phosphoacceptors, although some dual specificity kinases and phosphatases have also been described. The importance of phosphorylation cascades is reflected by the finding that many kinases, phosphatases, and the signal transduction pathways in which they participate have been highly conserved during the course of evolution. In recent years, interest has focused on the role of protein phosphorylation in the control of the cell cycle; a number of cellular proto-oncogenes encode members of the serine/threonine kinase family and it has become increasingly clear that certain serine/threonine kinases function as key components of the cell cycle regulatory network. Therefore, the complete delineation of these pathways is an important aim for the understanding of oncogenesis and tumour progression.
Loss of sensitivity to negative growth regulators may be an important step in the development of malignant tumours. For example, transforming growth factor beta (TGFbeta), a potent natural antiproliferative agent, is believed to play an important role in suppressing tumorigenicity. Comparisons of human colon carcinoma and melanoma cell lines have demonstrated a progressive loss of responsiveness to the growth inhibitory effects of TGFbeta as tumour aggressiveness increases (Filmus et al., 1993, Curr. Opin. Oncol., 5, 123-129; Roberts et al., 1993, Growth Factors, 8, 1-9).
It has become increasingly clear over the last 10 years that the products of most of the genes involved in cellular transformation and cancer i.e. oncogenes and tumour suppressor genes are components of signal transduction pathways (Hanahan and Weinberg, 2000, Cell, 100, 57-70). As a result of extensive studies of the PI-3K/PKB signal transduction pathway, PKB was determined to play a major role in PI-3K induced signalling and involved in the regulation of various aspects of cellular processes (Galetic et al., 1999, Pharmacol. Ther., 82, 409-425; Vanhaesebroeck and Alessi, 2000, Biochem. J. 346 Pt 3, 561-576). For example, PKB regulates cell survival by phosphorylating and either activating the anti-apoptotic factor BAD (Datta et al., 1997, Cell, 91, 231-241), or inhibiting the pro-apoptotic and growth suppressor Forkhead transcription factors FKHR, FKHRL1 and AFX (Brunet et al., 1999, Cell, 96, 857-868; Kops et al., 1999, Nature, 398, 630-634; Rena et al., 1999, J. Biol. Chem., 274, 17179-17183; Biggs et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 7421-7426; Guo et al., 1999, J. Biol. Chem., 274, 17184-17192; Medema et al., 2000, Nature, 404,782-787).
c-Akt/PKB is an ubiquitous Ser/Thr protein kinase which has a complex mechanism of regulation yet to be completely resolved (Downward et al., 1998, Curr. Opin. Cell Biol., 10, 262-267; Coffer et al., 1998, Biochem. J., 335, 1-13; Kandel and Hay, 1999, Exp. Cell Res., 253, 210-229; Vanhaesebroeck and Alessi, 2000). PKB is highly activated by the PI-3K-generated second messenger, phosphatidylinositol 3,4,5 phosphate (PI-3,4,5-P3), due to the presence at its N-terminus of a PH domain that has a high affinity for this lipid (Frech et al., 1997, J. Biol. Chem., 272, 8474-8481; Kavran et al., 1998, J. Biol. Chem., 273, 30497-30508). The use of the PI-3K specific inhibitors Wortmannin and LY294002 has clearly demonstrated that PKB, in-vivo, is a downstream target of PI-3K upon cell stimulation by a wide variety of stimuli such as PDGF, EGF, bFGF, serum, insulin and IGF-1 (Burgering and Coffer, 1995, Nature, 376, 599-602; Cross et al., 1995, Nature, 378, 785-789; Franke et al., 1995, Cell, 81, 727-736; Kohn et al., 1995, EMBO J., 14, 4288-4295; Alessi et al., 1996, EMBO J., 15, 6541-6551; Andjelkovic et al., 1996, Proc. Natl. Acad. Sci. USA, 93,5699-5704). These results suggested that PKB might be bound to the membrane for activation since PI-3,4,5-P3 is located in the plasma membrane. Indeed, after cell stimulation, PKB is effectively translocated from the cytoplasm to the membrane in a wortmannin-dependent manner (Kohn et al., 1996, J. Biol. Chem., 271, 21920-21926; Andjelkovic et al., 1997, J. Biol. Chem., 272, 31515-31524). The membrane localization of PKB for activation is further supported in that only a constitutively membrane-bound PKB, such as the retroviral oncogene v-Akt or a chimeric PKB with a myristoylation motif at its N-terminus are able to transform cells (Bellacosa et al., 1993, Oncogene, 8, 745-754; Aoki et al., 1998, Proc. Natl. Acad. Sci. USA, 95,14950-14955).
When bound to the plasma membrane, PKB is required to be phosphorylated on residues Thr308 (in the activation loop) and Ser473 (in the C-terminal regulatory domain) for full activation in a wortmannin-dependent manner (Alessi et al., 1996). In cells lacking the tumour suppressor PTEN (a lipid phosphatase), PKB is more active (Cantley and Neel, 1999, Proc. Natl. Acad. Sci. USA, 96, 4240-4245; Vazquez and Sellers, 2000, Biochim. Biophys. Acta, 1470, M21-M35), as a result of the increase in phosphorylation at these residues. The kinase PDK1, a kinase that contains a PH domain, has been shown to be able to phosphorylate PKB at Thr-308 in-vivo (Alessi et al., 1997, Curr. Biol., 7, 261-269; Stokoe et al., 1997, Science, 277, 567-570; Stephens et al., 1998, Science, 279, 710-714). Although PDK1 or even PKB itself can phosphorylate Ser473, the kinase responsible for Ser473 phosphorylation in vivo, often referred to as PDK2 or Ser473 kinase has not yet been identified (Balendran et al., 1999, Curr. Biol., 9, 393-404; Biondi et al., 2000, EMBO J., 19, 979-988; Toker and Newton, 2000, J. Biol. Chem., 275, 8271-8274).
The yeast two hybrid system (Fields and Song (1989) Nature 340, 245-246; Chien et al. (1991) Proc. Natl. Acad. Sci. USA 88, 9578-9582) has previously been used to determine if protein kinase B (PKB) could function by forming specific interactions with other proteins (PCT WO9718303). The existence of a PKB-interacting protein, Carboxy-Terminal Binding Protein (CTBP), was also described therein. For clarification, PKB was previously known as RAC-PK and AKT. Proteins and other factors that interact with PKB need to be identified to allow manipulation of PKB signalling pathways and their dependent cellular processes. The present invention relates to the identification of a novel PKB-interacting protein and its characterization as a tumour suppressor agent.